T-box genes encode a family of transcription factors that regulate morphogenesis and differentiation. The investigators present a new way to view T-box gene function that has significant implications for how to understand human malformations caused by T-box gene mutations. They propose that T-box genes can regulate each other's expression and activity, and hence the combined expression of T-box genes in a cell is crucial for determining its developmental fate. This hypothesis would account for the findings that T-box genes are often expressed in overlapping domains and that T-box genes often appear not to have a uniform function throughout their expression domain. To test this hypothesis, they will examine regulatory interactions between three zebrafish T-box genes expressed in the early mesoderm. Expression of the genes is initially nested: no tail is expressed throughout the mesoderm precursor population, although it has distinct functions in the dorsal and ventral mesoderm. tbx6 and spadetail (spt) are expressed within the ventral mesoderm. They propose that one function of tbx6 and spt in the ventral mesoderm is to suppress the expression of dorsal-specific genes that can be activated by no tail. They propose that the T-box genes interact because the transcription factors they encode can compete for regulation of some downstream genes. Aim #1 tests genetic interactions between the three genes. Ectopic expression is used to test if spt can suppress dorsal functions of no tail. tbx6 mutants are isolated and the effects of tbx6, spt, and tbx6;spt double mutants are analyzed. Aim #2 tests directly, using transactivation studies in vitro and reporter transgenes in embryos, whether the transcription factors can compete at defined binding sites for regulation of reporter genes. Aim #3 uses differential hybridization screening to identify endogenous gene targets of no tail that are expressed in a domain-specific manner. Promoter regions of target genes will be analyzed to determine the cis-regulatory sequences responsible for domain-specific expression